1. Field of the Invention
The present invention relates to a novel strain of microorganism, a method of biodegrading chemical compounds, such as trichloroethylene, phenolic compounds and furan compounds by using thereof, a method of remedying environment such as sewage, waste water, soil and so on. The present invention also relates to a method of producing 2-furan carboxylic acid from furfural biologically.
2. Related Background Art
In recent years, various environmental inspections have reported that harmful and less degradable aromatic chemical substances have been detected, and in consequence, much attention has now been paid to environmental pollution with these substances. The influence of these substances on ecological systems is feared.
Therefore, in order to prevent pollution with these less degradable chemical substances, it is required to rapidly develop a technique by which these substances are inhibited from getting into the environment. For example, it is strongly desired to establish a technique by which the less degradable harmful substances can be effectively removed from sewage or waste water. Furthermore, the pollution of a soil with the less degradable harmful substances not only hinders the reutilization of the soil but also causes further escalation of the pollution due to the running of the pollutant into groundwater, which is a serious social problem. Therefore, it is strongly desired to establish a technique by which the escalation of the pollution with the less degradable chemical substances can be prevented and the polluted environment can be remediated.
Examples of such non-decomposable substances include phenolic compounds such as phenol and cresol; furan compounds such as furfural, tetrahydrofuran, furfuryl alcohol, and cumaran.
Although phenol found in various waste liquids can be decomposed by a chemical decomposition method using light, heat, ozone or the like, microbial decomposition attracts attention from the viewpoints of treatment cost, and operation. Examples of microorganisms known as having the ability to decompose phenol include bacteria belonging to Pseudomonas, Nocardia, Bacillus, Acinetobacter, Aureobasidium; fungi belonging to Fusarium and the like; yeasts belonging to Triccosporon, Candida and the like. Typical examples of bacteria belonging to the Pseudomonas include Pseudomonas putida, and Pseudomonas paucimobilis. The Pseudomonas is now also known as Burkholderia.
Cresol is found in waste liquids of coal gasification factories, groundwater contaminated with gasoline, waste liquids of petroleum refineries and the like. The purification of such waste liquids by decomposing cresol becomes a critical problem from the viewpoint of environmental protection. Although cresol can also be decomposed by a chemical decomposition method using light, heat, ozone or the like, microbial decomposition is appreciated from the viewpoints of treatment cost and operation. However, there is substantially no isolated microorganism having the ability to decompose cresol, and only a few bacteria belonging to Pseudomonas such as Pseudomonas QT31 strain (C. Masque et al., Biotechnology Letters, Vol. 9, No. 9, 655-660, 1987) and the like are reported as cresol resistant strains.
There are only a few reports on isolated microorganisms having the ability to decompose furfural or tetrahydrofuran. Such reports include a report on decomposition of furfural by culturing bread yeasts (S. Morimoto et al., J. Ferment. Technol., 45, 442, 1967), and a former Soviet patent (Japanese Patent Publication No. 44-20389) on conversion of furfural to 2-furan carboxylic acid by Acetobacter, Achromobacter, Brevibacterium, Flavobacterium, Micrococcus or the like (refer to Conversion of Organic Compound by Microorganism, by G. K. Skryabin and L. A. Golovleva, translated by Saburo Fukui, Gakkai Shuppan Center, 287). An experiment performed under anaerobic conditions is described in Brune, G., Schovert h, S. M., and Sahm, H. (1982); Process Biochem., 17, 20. There is substantially no example of isolated microorganisms having the ability to decompose tetrahydrofuran or cumaran.
There is, thus, a great demand for practical reasons for obtaining microorganisms having the ability to decompose such non-decomposable furan compounds. As is common to aldehydes, furfural among these furan compounds has high toxicity. Thus, it is important to obtain microorganisms having resistance to the toxicity of furfural. In addition, in treatment of a compound having a cumaran ring as a basic skeleton thereof, microorganisms having the ability to decompose the cumaran ring is useful and is, therefor in great demand.
On the other hand, 2-furan carboxylic acid among the furan compounds has lower toxicity than that of furfural and is easily assimilated by microorganisms. It is to be expected that the conversion of furfural available at low cost and furan compounds mixed in various waste liquids to 2-furan carboxylic acid promotes the utilization of furan compounds in the fermentation industry.
In particular, the oxidative conversion of furfural to 2-furan carboxylic acid is a very significant means for decreasing the toxicity of furfural and introducing it as a raw material into the fermentation industry. Known methods of converting furfural to 2-furan carboxylic acid include various methods using chemical reactions. However, any one of the methods requires violent reaction, and produces high concentrations of various toxic substances in the final reaction solutions which inhibit the growth of microorganisms. When the 2-furan carboxylic acid obtained by the chemical method is used as a raw material for fermentation, it requires a troublesome operation of separating the toxic substances from the final reaction solution. From this viewpoint, attention is paid to a method of producing 2-furan carboxylic acid by using microorganisms to obtain a target substance under very mild conditions.
However, as described above, there are only a few reports on isolated microorganisms used for converting furfural to 2-furan carboxylic acid. There is, thus, a demand to obtain practical useful microorganisms.
Furfural can easily be obtained, for example, by steam distillation of a plant containing pentosan or treatment thereof with a mineral acid, and is industrially produced from hulls of oats or wheat straws. Of the furan compounds, furfural is available at the lowest cost and is widely used as a selective purification solvent for lubricating oil, a vulcanization accelerator, a dye penetrating agent and the like.
Tetrahydrofuran is useful as an organic solvent having excellent dissolving ability and is widely used as a solvent component for surface coating, an adhesive, a paint release agent and the like. Furfuryl alcohol is used as a solvent or a dispersant for dyes, phenolic resins, and a lubricant. A compound having a cumaran (2,3-dihydrobenzofuran) ring is widely used for production of dyes and pigments, as an organic solvent. The compound is also found as a component of wood.
The above compounds are thus mixed in waste liquids of various chemical factories, and the presence of tetrahydrofuran presents the danger of contaminating groundwater and the like because of water solubility. Accordingly, it is important from the viewpoint of environmental protection to decompose these compounds.
Although the non-decomposable compounds can be decomposed by a method using light, heat, ozone or the like, a biological decomposition method employing a microorganism is appreciated from the viewpoint of cost and operation properties.
There are known microorganisms having the ability to decompose phenolic compounds which include the above strains. However, satisfactory strains which satisfy practical conditions and have a sufficient decomposing ability when used for decomposing a phenolic compound using microorganisms are not found in the currently known strains. It is thus necessary to obtain a strain which satisfies the characteristics required for practical use.
In addition, only few microorganisms are known as microorganisms having the ability to decompose furan compounds which are not easily decomposed under natural conditions, and strains which satisfy practical conditions and have the sufficient decomposing ability are not currently known. Therefore, it is necessary to obtain a strain which satisfies the characteristics required for practical use.
Similarly, satisfactory microorganisms which are employed for producing 2-furan carboxylic acid from a furan compound are not found, and it is necessary to obtain a strain which satisfies the characteristics required for practical use.
The characteristics required for practical use are as follows: (1) A strain must have the ability to decompose a phenolic compound or a furan compound or the ability to convert a furfural compound to 2-furan carboxylic acid; (2) A broader range of growth conditions than those of known strains; (3) The applicability can be increased, or a strain can be utilized in various forms.
For example, microorganisms used for treating a waste water containing a phenolic compound or a furan compound are required not to be damaged easily in the waste water, necessitating microorganisms that can be grown under poor conditions, as in waste water. Microorganisms having resistance to many antibiotics and the ability to utilize various kinds of sugar have a greater possibility to grown well under poor environmental conditions. It is thus important to obtain microorganisms having resistance to various chemicals, as well as high utilization of sugar.
At an actual contamination site, composite contamination is frequently produced by several chemical substances rather than a single chemical substance. A treatment method using different kinds of bacteria for decomposing the respective chemical substances causes a problem with respect to differences in the growth conditions of the bacteria used and requires more complicated control. There is thus a demand for microorganisms of a single strain having the ability to decompose several chemical substances.
Another example of non-decomposable substance is trichloroethylene (TCE). TCE is a chlorinated organic compound which has been used in IC industries, dry cleaning and the like, and it is a carcinogen. Thus, the environmental pollution with TCE inclusive of the problem of the soil pollution caused by the pollution of groundwater is a serious social problem. Accordingly, the removal and the degradation of TCE contained in the environment, the purification of sewage or waste water containing TCE, and the remediation of the polluted soil are important themes from the viewpoint of environmental protection.
As a removal treatment and a degradation treatment of TCE, there are an adsorption treatment using active carbon, a degradation treatment utilizing light or heat, and the like. However, a biodegradation treatment using microorganisms is attracting attention from the standpoints of cost and operability.
There is a technique by which the function of microorganisms in a soil is utilized to degrade pollutants in the soil and to thereby eliminate the environmental pollution, and this technique is called bioremediation, because of remediating the soil by the use of the microorganisms. Hence, it can be expected that the bioremediation technique is applied to the remediation of polluted soils such as the vacant lot of a semiconductor manufacturing factory, a site of a metal processing factory, the vacant lot of a chemical plant, and the like.
However, there are not a many of reports that microorganisms having a TCE degrading ability have been isolated. Examples of the microorganisms having TCE degrading ability are limited, and they include Welchia alkenophila sero 5 (U.S. Pat. No. 4,877,736, ATCC53570), Welchia alkenophila sero 33 (U.S. Pat. No. 4,877,736, ATCC53571), Methylosinus trichosprium OB3b [Whitenbury R. J., Gen. Microbiol, Vol. 61, pp. 205-218 (1970)], Pseudomonas sp. G4 [Nelson M. J. K. et al., Appl. Eviron. Microbiol., Aug., pp. 383-384 (1986); Folsom B. R. et al., Appl. Eviron. Microbiol., May, pp. 1279-1285 (1990); and U.S. Pat. No. 4,925,802, ATCC53617; this bacterium has first belonged to Pseudomonas cepacia but then changed to Pseudomonas sp.], Methylomonas sp. MM2 [Henry S. M. et al., Appl. Environ. Microbiol., Jan., pp. 236-244 (1991)], Alcaligenes denitrificans ssp. Xylosoxidsans JE75 [Ewers J. et al., Arch. Microbiol., Vol. 154, pp. 410-413 (1990)], Alcaligenes eutrophus JMP 134 [Harker A. R. & Kim Y. Appl. Environ. Microbiol., Apr., pp. 1179-1181 (1990)], Pseudomonas putida F1 [Gibson DT et al., Biochem., Vol. 7, pp. 2653-2662 (1968); Wackett L. P. & Gibson D. T., Appl. Environ. Microbiol, July, pp. 1703-1708 (1988)], Mycobacterium vaccse JOB5 [Beam H. W. & Perry J. J., J. Gen. Microbiol., Vol. 82, pp. 163-169 (1974); Wackett L. P. et al., Appl. Environ. Microbiol., Nov., pp. 2960-2964 (1989), ATCC29678], Nitrosomonas europaea [Arciero D. et al., Biochem. Biophys. Res. Comm., Vol. 159, pp. 640-643 (1989)], Pseudomonas fluoescens PFL12 [Vandenbergh P. A. & Kunka B. S., Appl. Environ. Microbiol., Oct., pp. 2578-2579 (1988)], Lactobacillus fuctivorans RE [Kunkee, Int. J. Syst. Bact., Vol. 30, pp. 313-314 (1980), J. Appl. Bact., Vol. 34, pp. 541-545 (1971)], Lactobacillus vaginalis sp. nov. [Embley T. M. et al., Int. J. Syst. Bacteriol., Vol. 39, pp. 368-370 (1989), ATCC49540], and Methylosinus trichosprium (Japanese Patent Application Nos. 2-92274 and 3-292970).
In addition, none of the presently known bacteria can meet the practical requirements for the TCE degradation method using the microorganisms and do not possess the sufficient degrading ability.
Particularly in the case the microorganisms are used in the soil, it must be considered that the treatment is carried out in a specific environment, i.e. in the soil. The microorganisms to be used are required to have a sufficient TCE degrading activity and to effectively show this activity in the soil, but in the conventionally known bacteria, these points are not sufficient.
Nowadays, the acquisition of the bacteria which can meet practically necessary characteristics is strongly desired.
Such microorganisms are those which preferably have sufficient TCE degrading ability, are different from known bacteria in growth conditions and the like, can be applied to a wide range, are rich in utilizable morphology, and particularly can be effectively utilized in the specific environment of the soil. Examples of such additional requirements include drug resistance and an ability to utilize sucrose.
For example, in the case that the waste water containing TCE is treated, it is required that the microorganisms to be used have a TCE degrading ability, are scarcely damaged in the waste water, and can grow in the severe environment of the waste water. That is, the microorganisms having resistance to many antibiotics and assimilability to various saccharoses can probably successfully grow even in the severe environment.
Thus, the bacteria having the TCE degrading ability and more practically advantageous characteristics than the conventionally known bacteria are strongly required.
Furthermore, in biodegrading chlorinated organic compounds such as TCE, tetrachloroethylene (PCE) and dichloroethylene (DCE) in an environment of a polluted site such as a polluted soil, i.e., an open system, the density of the administered microorganisms by which the biodegradation can be carried out is noticeably decreased owing to predation by protozoans and under the influence of other native bacteria. For this reason, it is often very difficult to increase the density of the microorganisms in compliance with a required treatment ability. In order to increase the density of the microorganisms, there are a method which comprises feeding air to the soil, and a method which comprises forwarding a nutritious solution under pressure. However, although requiring a great deal of energy, these methods cannot effectively increase the number of the bacteria per unit area, and so the treatment ability of these methods remains at a low level on the whole.
In the treatment in a reactor or the like, i.e., in the treatment in a closed system, a good deal of energy for nutrient feed and aeration is also required so as to maintain the density of the microorganisms, as in the above-mentioned open system.
The microorganisms which can degrade the chlorinated organic compounds express an enzyme capable of degrading these compounds, but in order to express this kind of enzyme, an inducer is necessary.
In this case, when a large amount of the inducer is used, the degradation activity of the microorganisms increases, and this fact is known only in an example of tryptophan (WO 90/06901). However, detailed reports regrading the amount of the inducer to be used have not been present at all.